1. Field of the Invention
This invention relates to serum radioassays and, more particularly, to improved polymeric competitive protein binding adsorbents for use in such assays.
In recent years, radioassay techniques have made possible detection and quantification of circulating levels of serum components present in extremely minute concentrations in blood. Typically in such assays, an unknown quantity of a given serum component is made to compete with a known quantity of a radioactively labeled "marker" substance for a limited number of binding sites on a competitive protein binding adsorbent specific for determinants characteristic of both the unknown and its homologous marker. The resulting complex is isolated from unbound moieties and its radioactivity is measured. A difference between the radioactivity of that complex and one similarly derived from a standard whose content of the substance under assay is known can be directly related to the concentration of unknown in the serum sample assayed.
While a wide variety of specific adsorbent substances may be employed in radioassay procedures, including receptor and other specific binding proteins, e.g., B.sub.12 intrinsic factor and thyroxin binding globulin, radioassay is most commonly applied to the detection of substances specifically bound by circulating antibodies. In such "radioimmunoassays", an immunogen or hapten under assay competes with homologous marker for binding sites on the antibody, whose radioactivity is then measured. In this context the term "immunogen" denotes any protein, polypeptide, polysaccharide, mucopolysaccharide or other substance, whether or not normally foreign to the circulatory system, which reacts specifically with a given antibody to form an antigen-antibody complex. A "hapten" is a small molecule or portion thereof capable of specifically binding to a given antibody but, by reason of its size, incapable of generating antibodies unless conjugated to an immunogenic carrier.
2. Description of the Prior Art
The isolation of the antigen-antibody complex from the other reactants for the measurement of its radioactivity in radioimmunoassays has been effected in various ways.
According to one procedure employed in radioimmunoassay of digoxin and other sterol-like compounds, as described by Meade and Kleist in J. Lab. Clin. Med. 80:748, 1972, the free digoxin was separated from the antibody-bound digoxin by adsorption of the former to dextran-coated charcoal. It was found, however, that a rapid dissociation of the digoxin-antibody complex occurred upon removal of the free digoxin by the dextran-coated charcoal, and that good results therefore were dependent upon a rigidly fixed time of contact with charcoal for all samples. Meade and kleist therefore investigated various methods of precipitating the antibody-bound digoxin to effect irreversible removal of the bound fraction.
Another approach, described by Donini and Donini in Karolinska Symposia, Stockholm, 1969, page 257, employed specific antisera made insoluble by polymerization for the separation of bound and free hormone in the radioimmunoassay of follicle stimulating hormone and luteinizing hormone. In their method, antisera were precipitated from aqueous solutions by the addition of ethyl chloroformate according to a modification of a procedure described by Avrameas & Ternynck in J. Biol. Chem., 242:1651 (1967).
According to Donini and Donini, the antisera were sometimes copolymerized with bovine serum albumin (BSA); this procedure being adopted when only a small amount of antiserum was available. For example, 0.2 to 0.5 ml of antiserum was brought to 2 ml by the addition of an appropriate amount of acetate buffer, pH 5.0, containing BSA in such a quantity that the final protein concentration amounted to 35 to 50 mg/ml. According to the authors, this copolymerization procedure was feasible only when the antiserum was used at a high dilution.